During glycolysis, cells convert simple sugars, such as glucose, into pyruvic acid, with a net production of ATP and NADH. In the absence of a functioning electron transport system for oxidative phosphorylation, at least 95% of the pyruvic acid is consumed in short pathways which regenerate NAD.sup.+, an obligate requirement for continued glycolysis and ATP production. The waste products of these NAD.sup.+ regeneration systems are commonly referred to as fermentation products (Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston [1987] "Genetic Engineering of Ethanol Production in Escherichia coli," App. and Env. Microbiol. 53:2420-2425).
Microorganisms are particularly diverse in the array of fermentations products which are produced by different genera (Krieg, N. R., and J. G. Holt, eds. [1984] Bergey's manual of systematic bacteriology. The Williams & Wilkins Co., Baltimore). These products include organic acids, such as lactate, acetate, succinate, and butyrate, as well as neutral products, such as ethanol, butanol, acetone, and butanediol. Indeed, the diversity of fermentation products from bacteria has led to their use as a primary determinant in taxonomy (Krieg and Holt [1984], supra).
End products of fermentation share several fundamental features. They are relatively nontoxic under the conditions in which they are initially produced but become more toxic upon accumulation. They are more reduced than pyruvate because their immediate precursors have served as terminal electron acceptors during glycolysis. The microbial production of these fermentation products forms the basis for our oldest and most economically successful applications of biotechnology and includes dairy products, meats, beverages, and fuels. In recent years, many advances have been made in the field of biotechnology as a result of new technologies which enable researchers to selectively alter the genetic makeup of some microorganisms.
Escherichia coli is an important vehicle for the cloning and modification of genes for biotechnology and is one of the most important hosts for the production of recombinant products. In recent years, the range of hosts used for recombinant DNA research has been extended to include a variety of bacteria, yeasts, fungi, and some eukaryotic cells. The invention described here relates to the use of recombinant DNA technology to elicit the production of specific useful products by a modified host.
The DNA used to modify the host of the subject invention is isolated from Zymononas mobilis. Z. mobilis is a microorganism with unusual metabolic characteristics which is commonly found in plant saps and in honey. Z. mobilis has long served as a natural inocula for the fermentation of the Agave sap to produce pulque (an alcohol-containing Mexican beverage) and as inocula for palm wines. This organism is also used for fuel ethanol production and has been reported capable of ethanol production rates which are substantially higher than that of yeasts.
Although Z. mobilis is nutritionally simple and capable of synthesizing amino acids, nucleotides and vitamins, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose. Substrate level phosphorylation from the fermentation of these sugars is the sole source of energy for biosynthesis and homeostasis. Z. mobilis is incapable of growth even in rich medium such as nutrient broth without a fermentable sugar.
Z. mobilis is an obligately fermentative bacterium which lacks a functional system for oxidative phosphorylation. This organism, like Saccharomyces cerevisiae, produces ethanol and carbon dioxide as principal fermentation products. Z. mobilis produces ethanol by a short pathway which requires only two enzymatic activities: pyruvate decarboxylase and alcohol dehydrogenase. Pyruvate decarboxylase is the key enzyme in this pathway which diverts the flow of pyruvate to ethanol. Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Two alcohol dehydrogenase isozymes are present in this organism and catalyze the reduction of acetaldehyde to ethanol during fermentation, accompanied by the oxidation of NADH to NAD.sup.+. Although bacterial alcohol dehydrogenases are common in many organisms, few bacteria have pyruvate decarboxylase. Attempts to modify Z. mobilis to enhance its commercial utility as an ethanol producer have met with very limited success.
The gene coding for pyruvate decarboxylase in Z. mobilis has been cloned, characterized, and expressed in E. coli (Brau, B., and H. Sahm [1986] "Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization," Arch. Microbiol. 146:105-110; Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and L. O. Ingram [1987] "Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase," J. Bacteriol. 169:949-954). The subject invention relates to the creation and expression of a novel system coding for the production of ethanol.